Immunological detection method and kit for mycoplasma pneumoniae

ABSTRACT

The present invention aims at providing a specific antibody that can simply and rapidly detect  Mycoplasma pneumoniae  which is a causative bacterium of  mycoplasma  pneumonia, with high sensitivity, and also an immunological detection method and a kit containing the same antibody. The present invention makes it possible to diagnose infection with  Mycoplasma pneumoniae  more rapidly and specifically than the conventional method, by producing an antibody recognizing a specific epitope of P30 protein of  Mycoplasma pneumoniae  and performing an immunological detection using the antibody. Also, the present invention enables easy and rapid detection of  Mycoplasma pneumoniae  and diagnosis of infection with the same at a hospital or the like without need of specialized instruments or skilled techniques.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/546,347, filed Jul. 28, 2017, as the U.S. national phaseapplication filed under 35 U.S.C. § 371 of International Application No.PCT/JP2016/052381, filed Jan. 27, 2016, designating the United States,which claims priority from Japanese Patent Application No. 2015-015253,filed Jan. 29, 2015, and the complete disclosures of such applications,including sequence listing(s), are hereby incorporated herein byreference in their entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on May 3, 2023, isnamed 7378156657US_SL.xml and is 7,608 bytes in size.

TECHNICAL FIELD

The present invention relates to an antibody against P30 protein ofMycoplasma pneumoniae, and an immunological method and a kit fordetecting Mycoplasma pneumoniae using the antibody.

BACKGROUND ART

Mycoplasma pneumonia is atypical pneumonia caused by Mycoplasmapneumoniae. Mycoplasma pneumonia, together with chlamydia pneumonia,constitutes 30% to 40% of the atypical pneumonia cases and alsoconstitutes a high percentage of the community-acquired pneumonia cases.

Mycoplasma pneumonia is common in infants, children, and adolescence.The incubation period is 2 to 3 weeks. Excretion of the pathogen intorespiratory mucosa is observed in 2 to 8 days before the onset ofinitial symptom, becomes the highest at the onset of clinical symptoms,continues at a high level for about one week, and then further continuesfor 4 to 6 weeks or more. Main clinical symptoms are fever, generalmalaise, headache, and other cold-like symptoms. Mycoplasma pneumonia ischaracterized by, for example, high fever greater than 38° C. andintense dry cough. The cough further continues for a long time, 3 to 4weeks, after decline of fever. However, there is no examination findingcharacteristic to mycoplasma pneumonia, and pale ground-glass appearancein chest X-ray examination is typical.

The manner of infection with Mycoplasma is droplet infection and contactinfection from an infected patient. Mycoplasma pneumoniae invades therespiratory tract and attaches to the bronchi or, bronchiole epitheliumto achieve the infection.

Mycoplasma infection is designated as a notifiable infectious disease(Infectious diseases Category V) based on the Infectious DiseasesControl Law, and designated medical care providers have an obligation topromptly report the number of patients.

Mycoplasma pneumoniae is a minimum microorganism that can replicateitself, and differs from other bacteria in that it does not have a cellwall. Accordingly, β-lactam antibiotics and cephem antibiotics, whichare antibiotics having a function of inhibiting cell wall synthesis, areineffective, and macrolide antibiotics, tetracycline antibiotics and newquinolone antibiotics are administered for treatment. Promptidentification of the causative bacteria is therefore desired fordetermining a treatment plan.

At present, Mycoplasma pneumoniae infection is definitely diagnosed byan isolation culture method and a serological test.

The isolation culture needs a specialized culture medium (PPLO medium)for detecting Mycoplasma. In addition, its proliferation is slow,compared to other bacteria, thereby taking at least about one week forobtaining the result of determination. It is therefore difficult torapidly identify the causative bacteria by the isolation culture methodin clinical sites.

Mycoplasma is susceptible to temperature, and samples containingMycoplasma cannot be kept in cold storage, unlike samples containingcommon bacteria. Accordingly, Mycoplasma contained in a sample maybecome extinct or decrease during storage or transportation of thesample and may not be detected even by the isolation culture method.

Examples of the serological test include a cold agglutination, aComplement Fixation (CF) test, an indirect hemagglutination (IHA) test,a particle agglutination (PA) method, and an enzyme immunoassay (EIA),which specifically detect IgG antibody or IgM antibody.

Furthermore, an immunochromatographic kit (ImmunoCard MycoplasmaAntibody, available from TFB, Inc.) is commercially available as asimple test detecting Mycoplasma pneumoniae-specific IgM antibody inserum or plasma by EIA and is used at clinical sites.

In the serological test, although the IgM antibody in a sample to bedetected increases at the early stage of infection, the sample may showfalse negative in the case of low antibody production response ordepending on assay timing. Furthermore, since it takes a long timebefore IgM antibody disappears in blood, it cannot be said that theresult of the serological test always correctly indicates the currentinfection status.

Accordingly, definite diagnosis by the serological test needsquantitative tests using paired sera of acute and convalescent stages,and therefore has to be ex-post diagnosis in many cases.

A nucleic acid detection method for detecting DNA of Mycoplasmapneumoniae is also employed. In the nucleic acid detection method,however, the procedure of amplifying nucleic acid is complicated andneeds specialized equipment, and the assay takes several hours. Thus,the method is not a test that is generally used.

In order to more rapidly and simply detect Mycoplasma pneumoniaeinfection, a specific antibody against Mycoplasma pneumoniae antigen hasbeen developed, and a detection method of distinguishing whethermycoplasma infection is present or not has been reported.

Mycoplasma pneumoniae attaches itself to the respiratory epithelial cellcilia with its adhesive organ in a form of a flask-shaped protrusion,and then moves to the cell surface by gliding motility and adheresthereto to achieve the infection. Production of an antibody specific toP1 protein (169 KDa), which is known as adhesive protein playing acentral role in this adhesion or gliding motility, and a detectionmethod using the P1 protein as a detection marker have been reported(Patent Documents 1 and 2).

It is also known that P1 protein, the antigen to be detected reported inthe above reports, has two genotypes and that the amino acid sequencescorresponding to the genotypes of P1 protein are different from eachother. Accordingly, in order to broadly detect Mycoplasma pneumoniae,production of each antibody against P1 protein of each P1 genotype or anantibody recognizing a common site of P1 protein of the differentgenotypes is necessary. In addition, seasonal epidemic has been reportedsuch that a genotype different from the epidemic genotype in the latestseason is detected, that is, the genotype changes depending uponepidemic seasons. Therefore, it is necessary to find out the genotype atan early stage of the epidemic and use an antibody specific thereto.

A detection method using DnaK protein, which is known to be conservedamong isolated strains of Mycoplasma pneumoniae compared with P1protein, as a detection marker (Patent Document 3) has been alsoreported. DnaK protein is also possessed by Mycoplasma genitaliumcausing human urinary infectious diseases, and therefore also showscross reactivity. Consequently, Mycoplasma pneumoniae cannot bespecifically detected by using the above protein as a detection marker.

Also, there is a report of mutant strains related to P30 protein (30kDa) which is an adhesion protein of Mycoplasma pneumoniae (Non-PatentDocument 1). In this report, SDS-PAGE detection method using a rabbitantisera against P30 protein has been reported. A polypeptide havingN-terminal amino acid sequence (102-181) and a polypeptide havingC-terminal amino acid sequence (139-275) of P30 protein are expressed asa fusion protein with modified murine dihydrofolate reductase (DHFR) toproduce rabbit antisera against the fusion protein. In this report, onlythe rabbit antisera having reactivity with P30 protein is disclosed, butno antibody recognizing a specific epitope in P30 protein is disclosed.Furthermore, it is unknown whether the antiserum can be used forimmunological detection in the present invention, and is unclear whetherit has a high specificity and reactivity.

If mycoplasma pneumonia is not appropriately treated, the symptoms maybe protracted or become severe or may further cause the spread of theinfection due to secondary infection. Accordingly, in order to selectappropriate treatment and antibiotics, rapid and conclusive detection ofMycoplasma pneumoniae is demanded.

Furthermore, even though rapid detection of Mycoplasma pneumoniae hasbeen contemplated, an antibody that can further specifically detectMycoplasma pneumoniae, and also an immunoassay and a kit containing suchan antibody have been demanded.

CONVENTIONAL TECHNICAL DOCUMENTS Patent Documents

-   Patent Document 1: Japanese Patent Laid-Open No. H5-304990-   Patent Document 2: Japanese Patent Laid-Open No. 2013-72663-   Patent Document 3: International Publication No. WO2011/068189

Non-Patent Documents

-   Non Patent Document 1: G. Layh-Schmitt et al., “The adhesin related    30-kDa protein of Mycoplasma peumoniae exhibits size and antigen    variability.”, FEMS Microbiology Letters, 152, 1997, p. 101-108.

SUMMARY OF INVENTION Problem to be Solved by the Invention

The present invention aims at specifically detecting the P30 protein ofMycoplasma pneumoniae in biological samples, thereby enabling highlyaccurate diagnosis of infection with Mycoplasma pneumoniae than before.

Means for Solving the Problem

The present inventors have succeeded in obtaining an antibody against aspecific epitope of the P30 protein by immunizing a mouse using the P30protein of Mycoplasma pneumoniae as an immunogen, and have found outthat Mycoplasma pneumoniae can be detected more specifically with highersensitivity than before by using the antibody in an immunoassay,especially a sandwich immunoassay, and particularly animmunochromatographic assay. Thus, the present invention has beencompleted.

That is, according to one aspect of the present invention, there isprovided a method of detecting Mycoplasma pneumoniae, comprising animmunoassay using an antibody against P30 protein of Mycoplasmapneumoniae, the antibody being an antibody against an epitope located inany one of amino acid sequences of SEQ ID NOS: 3 to 5.

Similarly, there is provided an immunoassay kit for Mycoplasmapneumoniae, comprising at least an antibody against P30 protein ofMycoplasma pneumoniae, the antibody being an antibody against an epitopeof the P30 protein located in any one of amino acid sequences of SEQ IDNOS: 3 to 5.

In particular, the immunoassay in the above detection method andimmunoassay kit is preferably a sandwich immunoassay such as anenzyme-linked immunosorbent assay (ELISA) and an immunochromatographicassay.

Therefore, according to another aspect of the present invention, thereis provided a method of detecting Mycoplasma pneumoniae, comprising asandwich immunoassay using first and second antibodies against P30protein of Mycoplasma pneumoniae, wherein at least one of the first andsecond antibodies is an antibody against an epitope located in any oneof amino acid sequences of SEQ ID NOS 3 to 5.

Similarly, there is provided a sandwich immunoassay kit for Mycoplasmapneumoniae, comprising at least first and second antibodies against P30protein of Mycoplasma pneumoniae, wherein at least one of the first andsecond antibodies is an antibody against an epitope of the P30 proteinlocated in any one of amino acid sequences of SEQ ID NOS: 3 to 5.

According to a preferred embodiment of the present invention, there isprovided an immunochromatographic assay for detecting Mycoplasmapneumoniae, comprising:

-   -   providing a membrane carrier having a capturing zone which is        formed by previously immobilizing a first antibody against P30        protein of Mycoplasma pneumoniae at a predetermined position;    -   chromatographically developing a liquid mixture in the membrane        carrier toward the capturing zone, said liquid mixture        containing a second antibody against the P30 protein and a        predetermined amount of a test sample, whereby a complex of an        antigen contained in the test sample and the second antibody is        captured by the capturing zone,    -   wherein at least one of the first and second antibodies is an        antibody against an epitope located in any one of amino acid        sequences of SEQ ID NOS: 3 to 5.

According to another preferred embodiment of the present invention,there is provided a Mycoplasma pneumoniae-detectingimmunochromatographic test strip, comprising at least first and secondantibodies against P30 protein of Mycoplasma pneumoniae and a membranecarrier, wherein the first antibody is previously immobilized at apredetermined position of the membrane carrier so as to form a capturingzone; and the second antibody is labeled with an appropriate labelingsubstance and is provided at a position separated from the capturingzone so as to be chromatographically developed in the membrane carrier,wherein at least one of the first and second antibodies is an antibodyagainst an epitope located in any one of amino acid sequences of SEQ IDNOS: 3 to 5.

Although the antibody against the P30 protein, which is essentially usedin the present invention and is an antibody against an epitope locatedin any one of amino acid sequences of SEQ ID NOS: 3 to 5, may be apolyclonal antibody or a monoclonal antibody, preferred is a monoclonalantibody from the viewpoint of reaction specificity.

The amino acid sequences of SEQ ID NOS: 3 to 5 constitute parts of theentire amino acid sequence of the P30 protein set forth in SEQ ID NO: 1and are regions containing an epitope of the P30 protein.

In a sandwich immunoassay such as immunochromatographic assay, althoughthe first and second antibodies used therein may be each a polyclonalantibody or a monoclonal antibody, from the viewpoint of reactionspecificity, generally speaking, at least one of the antibodies ispreferably a monoclonal antibody, and both antibodies are particularlypreferably monoclonal antibodies. In addition, a large number of P30protein molecules are present and are localized on the cell surface. Inorder to avoid competition between antibodies used therein and to obtainhigher reactivity, the first antibody and the second antibody arepreferably antibodies against different epitopes of P30 protein.

Incidentally, the P30 protein of Mycoplasma pneumoniae to which theantibody for detecting Mycoplasma pneumoniae according to the presentinvention reacts is a protein necessary for adhesion of Mycoplasma to ahost cell and is known as one of the accessory proteins that worktogether with an adhesion factor P1 protein.

The P30 protein has a molecular weight of 30 KDa and is one of theadhesive proteins involved in adhesion and pathogenicity, like P1protein. In the Mycoplasma pneumoniae cell, the P30 protein is localizedon the cell surface at an end of the adhesive organ and is atransmembrane protein having the N-terminal region embedded in the cellmembrane and the C-terminal region present outside the cell membrane.The P30 protein includes an amino acid sequence containing a largenumber of proline on the C-terminal region and has a repeating structureof such a sequence. In general, a region having an amino acid sequencecontaining a large number of proline is known to form athree-dimensional conformation and is known to have a high possibilityof becoming an epitope reactive with an antibody.

The antibody used in the present invention is an antibody against anepitope located in any one of amino acid sequences of SEQ ID NOS: 3 to 5that are contained in the whole amino acid sequence of the P30 proteinshown in SEQ ID NO: 1. Also, such antibodies react with Mycoplasmapneumoniae only, but do not react with any other Mycoplasma bacteria,and thus are excellent in specificity. The amino acid sequences of SEQID NOS: 3 to 5 are each a region including an epitope of P30 protein.Therefore, the antibodies used in the present invention can be eachparaphrased as an antibody which can cause antigen-antibody reactionwith a fragment of P30 protein having 12 to 15 amino acid residuescontained in any one of amino acid sequences of SEQ ID NOS: 3 to 5.

Thus, according to another aspect of the present invention, there isprovided an antibody recognizing an epitope of P30 protein located inany one of amino acid sequences of SEQ ID NOS: 3 to 5.

Effect of Invention

According to the present invention, infection with Mycoplasma pneumoniaecan be rapidly and specifically diagnosed by producing an antibodyspecifically reactive to Mycoplasma pneumoniae P30 protein andperforming an immunological assay using the P30 protein as a detectionmarker. The immunological assay and assay apparatus of the presentinvention enables the diagnosis of infection with Mycoplasma pneumoniaeto be made more simply and rapidly than before, at a hospital or anotherfacility without need of any specialized equipment or skill.

According to the present invention, an antibody against an epitopelocated in any one of the amino acid sequences of SEQ ID NOS: 3 to 5contained in the whole amino acid sequence of P30 protein shown in SEQID NO: 1 is used in the immunoassay-based detection method, and thus thediagnosis of infection with Mycoplasma pneumoniae can be performed withhigher accuracy than before, and the detection can be made with highersensitivity at an earlier stage.

BRIEF DESCRIPTION OF DRAWING

The FIGURE is in two parts: FIG. 1A is a plan view of animmunochromatographic test strip, and FIG. 1B is a verticalcross-sectional view of the immunochromatographic test strip shown inFIG. 1A.

DESCRIPTION OF EMBODIMENTS

In the present invention, each step in production of an antibody anddetection or assay method using the antibody is performed in conformitywith each immunological procedure known per se.

In the present invention, a polyclonal antibody can be obtained, forexample, by cloning a DNA fragment corresponding to an amino acidsequence of SEQ ID NOS: 3 to 5 from the DNA sequence encoding the aminoacid sequence shown in SEQ ID NO: 1, allowing the cloned gene to expressin a host such as Escherichia coli in a genetic engineering manner,extracting and purifying the expressed protein, and immunizing an animalwith the purified protein being used as an antigen according to anordinary method, and then obtaining the polyclonal antibody from theantiserum of the immunized animal.

In the present invention, a monoclonal antibody can be obtained, forexample, by immunizing an animal such as a mouse with the above purifiedprotein being used as an antigen, fusing the splenic cells of theimmunized animal with myeloma cells for cell fusion, selecting the thusfused cells in a HAT-containing medium and allowing them to grow, andthen selecting the grown strains using the above polypeptide of an aminoacid sequence of SEQ NOS: 3 to 5 by an enzyme-labeled immunoassay or thelike.

Alternatively, the monoclonal antibody can be obtained, for example, bypurifying P30 protein from Mycoplasma pneumoniae, immunizing an animalsuch as a mouse with the P30 protein being used as an antigen, fusingthe splenic cells of the immunized animal with myeloma cells for cellfusion, selecting the thus fused cells in a HAT-containing medium andallowing them to grow, and then selecting a strain reactive with apolypeptide of SEQ ID NOS: 3 to 5 from the grown strains.

Examples of the antibody of the present invention include not onlyantibodies but also antibody fragments and modified antibodiessubstantially equivalent to the antibodies as having a reactivity with apolypeptide of an amino acid sequence of SEQ ID NOS: 3 to 5 contained inthe P30 protein of Mycoplasma pneumoniae. Examples of the antibodyfragments include Fab fragments, F(ab′)2 fragments, Fab′ fragments, andscFv fragments.

The immunochromatographic assay of the present invention for detectingMycoplasma pneumoniae in a test sample can be practiced easily inaccordance with the structure of a known immunochromatographic teststrip.

Generally, such an immunochromatographic test strip is constituted by atleast a first antibody which is capable of undergoing antigen-antibodyreaction at a first antigenic determinant of an antigen, a secondantibody which is labeled and capable of undergoing antigen-antibodyreaction at a second antigenic determinant of the antigen, and amembrane carrier, wherein the first antibody is previously immobilizedin a predetermined position of the membrane carrier so as to form acapturing zone, and the second antibody is placed at a positionseparated from the capturing zone so as to be allowed to bechromatographically developed in the membrane carrier. Although thefirst antibody and the second antibody may be each a polyclonal antibodyor a monoclonal antibody as described above, at least one of them ispreferably a monoclonal antibody. The first antibody and the secondantibody are generally used in a “hetero” combination. That is, thefirst and second antibodies which recognize the respective antigenicdeterminants different in both position and conformation on an antigenare used in combination. However, the first antigenic determinant andthe second antigenic determinant may have the same conformation as longas they are different in position on the antigen, and in such a case,the first antibody and the second antibody may be monoclonal antibodiesin a “homo” combination, that is, one and the same monoclonal antibodycan be used as both the first antibody and the second antibody.

As a specific example, mention may be made of a test strip as shown inFIGS. 1A and 1B. In FIGS. 1A and 1B, the numeral 1 indicates an adhesivesheet, 2 indicates an impregnated member, 3 indicates a membranecarrier, 31 indicates a capturing zone, 32 indicates a control capturingzone, 4 indicates an absorbing member, and 5 indicates asample-receiving member.

In the example shown in the drawing, the membrane carrier 3 consists ofan elongated strip-shaped nitrocellulose membrane filter having a widthof 5 mm and a length of 36 mm.

In the membrane carrier 3, a first antibody is immobilized at a positionof 7.5 mm from the end on the starting side of chromatographicdevelopment, so as to form a capturing zone 31 of an analyte.Furthermore, the membrane carrier 3 is provided with a control capturingzone 32 at a position of 15 mm from the end on the starting side ofchromatographic development. This control capturing zone 32 is providedfor verifying whether the reaction is performed or not regardless of thepresence or absence of an analyte and can be usually formed byimmobilizing a material (excluding the analyte) specificallyimmunologically binding to the second antibody to the membrane carrier3. For example, when an antibody derived from a mouse is used as thesecond antibody, an antibody against the mouse antibody can be used.

In the example shown in the FIGURE, a nitrocellulose membrane filter isused as the membrane carrier 3. However, any type of membrane carriercan be used herein, as long as it is able to chromatographically developan analyte contained in a test sample and immobilize an antibody thatforms the capturing zone 31. Thus, other types of cellulose membranes,nylon membranes, glass fiber membranes, or the like can also be used.

The impregnation member 2 comprises a member impregnated with a secondantibody that undergoes antigen-antibody reaction with the antigen at asecond antigenic determinant located at a site different from the firstantigenic determinant to which the first antibody binds. The secondantibody is previously labeled with an appropriate labeling substance.

In the example as shown in the FIGURE, a strip-shaped glass fibernonwoven fabric having a size of 5 mm×15 mm is used as the impregnatedmember 2. However, the impregnated member 2 is not limited thereto, butincludes, for example, cellulose fabrics (a filter paper, anitrocellulose membrane, etc.), porous plastic fabrics such as ofpolyethylene and polypropylene, and others.

As a labeling substance that labels the second antibody, any substancecan be used, as long as it is usable herein. Examples of such a labelingsubstance include a color labeling substance, an enzyme labelingsubstance, a fluorescent labeling substance, and a radiation labelingsubstance. Of these, a color labeling substance is preferably usedbecause observation of a color change in the capturing zone 31 withnaked eyes enables rapid and simple determination.

Examples of the color labeling substance include colloidal metals, suchas colloidal gold and colloidal platinum; and latexes, such as syntheticlatexes such as polystyrene latexes colored with pigments such as redand blue pigments and natural rubber latexes. Among these, colloidalmetals, such as colloidal gold, are particularly preferred.

The impregnation member 2 can be produced, for example, by impregnatinga member, such as the above-mentioned glass fiber non-woven fabric, witha suspension of a labeled second antibody and drying it.

As shown in FIGS. 1A and 1B, the immunochromatographic test strip of thepresent invention can be produced as follows. The membrane carrier 3 isaffixed to the middle of the adhesive sheet 1. On the end on thestarting side of chromatographic development (that is, the left side inFIGS. 1A and 1B which is hereinafter referred to as an “upstream side”whilst the opposite side, that is, the right side in FIGS. 1A and 1B ishereinafter referred to as a “downstream side”) of the membrane carrier3, the downstream side end of the impregnated member 2 is laid so as tocommunicate them. The upstream side zone of the impregnated member 2 isaffixed to the adhesive sheet 1.

Moreover, if necessary, the downstream side zone of a sample-receivingmember 5 may be placed on the upper face of the impregnated member 2whilst the upstream side zone of the sample-receiving member 5 may alsobe affixed to the adhesive sheet 1. Furthermore, the upstream side zoneof an absorbing member 4 may be placed on the upper face of thedownstream side zone of the membrane carrier 3 whilst the downstreamside zone of the absorbing member 4 may be affixed to the adhesive sheet1.

The absorbing member 4 may be made of any material as long as it is ableto quickly absorb and retain a liquid. Examples of such a materialinclude cotton fabrics, filter paper, and porous plastic nonwovenfabrics made from polyethylene, polypropylene, etc. In particular,filter paper is optimal. Also, a filter paper made of a compositematerial containing a water absorptive polymer may be used.

As the sample-receiving member 5, may be used, for example, a sheet orfilm of a porous synthetic resin such as porous polyethylene and porouspolypropylene, or cellulose paper or a woven or nonwoven fabric such asa filter paper and a cotton fabric.

Furthermore, the immunochromatographic test strip shown in FIGS. 1A and1B can be provided in a state of being accommodated in an appropriateplastic case having a test sample injection portion and a determinationportion which are respectively opened above the sample-receiving member5 and the capturing zone 31. In order to prevent secondary infection ofa user, the immunochromatographic test strip is preferably provided in astate being accommodated in the plastic case.

Then, a test sample constituted by a biological sample or the like is,if required, mixed with a suitable developing solvent so as to obtain aliquid mixture that can be developed chromatographically. Thereafter,the liquid mixture is injected into the sample-receiving member 5 of theimmunochromatographic test strip as shown in FIG. 1 , so that it passesthrough the sample-receiving member 5 and is mixed with a labeled secondantibody at the impregnated member 2.

In this instance, if an analyte exists in the aforementioned liquidmixture, a complex of the analyte and the second antibody is formed as aresult of antigen-antibody reaction. This complex is developedchromatographically in the membrane carrier 3, and then reaches thecapturing zone 31 and is captured by the first antibody immobilizedtherein as a result of antigen-antibody reaction.

In this instance, if a color labeling substance such as colloidal goldis used as a labeling substance, the analyte can be immediatelydetermined qualitatively or quantitatively based on coloring caused byaccumulation of the color labeling substance at the capturing zone 31.Furthermore, the intensity of the coloring can be digitized and can bequantitatively measured by optically reading the intensity of thecoloring of the color labeling substance accumulated on the capturingzone 31 of the immunochromatographic test strip with animmunochromatography reader.

In addition, when the chromatographic development is normally performed,the second antibody not involved in the antigen-antibody reaction withthe analyte reaches the control capturing zone 32 and is captured by anantibody that is immobilized therein and reactive to the secondantibody. On this occasion, if a color labeling substance is used as alabeling substance, the control capturing zone 32 colors by accumulationof the color labeling substance to confirm that the chromatographicdevelopment has been normally performed. In contrast, if the controlcapturing zone 32 does not color, it indicates that a problem such as nodevelopment of the second antibody happens.

Any test sample can be used. For example, it may be a biological samplein which Mycoplasma pneumoniae may be present, such as nasal cavityaspirate, nasopharynx aspirate, nasal cavity swab, throat swab,nasopharynx swab, sputum, saliva, and bronchial washings. The testsample may be diluted with an appropriate diluent such as physiologicalsaline and developing solvent before it is applied to the membranecarrier.

When a test sample contaminated with blood is used in a test, inparticular, using an antibody labeled with a color labeling substancesuch as colloidal gold, a hematocyte-capturing membrane member ispreferably disposed on the sample receiving member. Thehematocyte-capturing membrane member is preferably laminated between theaforementioned impregnated member and the aforementionedsample-receiving member. This inhibits development of erythrocytes inthe membrane carrier and thus facilitates the confirmation ofaccumulation of color labeling substances in the capturing zone of themembrane carrier. As such a hematocyte-capturing membrane member, acarboxymethyl cellulose membrane is used. Specifically, an ion exchangefilter paper CM (trade name) available from Advantec Toyo K.K., an ionexchange cellulose paper available from Whatman Japan K.K., etc. can beused.

EXAMPLES

The present invention will be described more specifically by way of thefollowing examples, but is not limited to the examples.

Example 1: Expression and Purification of Recombinant P30 Protein

The amino acid sequence of P30 protein of Mycoplasma pneumoniae M129strain was obtained from a database of DNA Data Bank of Japan (DDBJ). Anextracellular region excluding a transmembrane domain, the amino acidsequence (AA74-274) set forth in SEQ ID NO: 2, was specified from theamino acid sequence of the P30 protein, and a gene sequencecorresponding to the amino acid sequence was synthesized. A His-tagexpression vector, pET302/NT-His, was cleaved with a restriction enzyme,EcoRI, was then dephosphorylated using an alkaline phosphatase, and wasmixed with the gene sequence, followed by a ligation reaction using DNALigation Kit Ver. 2 (Takara Bio Inc.). The recombinant P30 plasmidcarrying the target gene was introduced into a recombinantprotein-expressing host, E. coli BL (DE3) pLysS (Novagen). The hostbacteria were cultured on an LB agar plate medium. The resultingcolonies were cultured in an LB liquid medium. Subsequently, 1 mM IPTG(Takara Bio Inc.) was added to the medium to induce expression ofrecombinant P30 protein, and E. coli was then collected. The collectedbacteria were resuspended in a solubilization buffer (0.5% Triron X-100(Sigma), 10 mM imidazole, 20 mM phosphate, and 0.5 M NaCl (pH 7.4)(Amersham)) and were solubilized by ultrasonication. The recombinant P30protein was then purified with His trap Kit (Amersham). This purifiedprotein was dialyzed against a phosphate buffered saline (hereinafter,referred to as PBS) to obtain a target recombinant P30 protein.

Example 2: Production of Monoclonal Antibody Against Recombinant P30Protein

The recombinant P30 protein prepared in Example 1 was used as an antigenfor immunization to produce a monoclonal antibody against therecombinant P30 protein (hereinafter, referred to as anti-P30 antibody).The monoclonal antibody was produced in accordance with an ordinarymethod. The recombinant P30 protein (100 μg) was mixed with an equalamount of Complete Freund's Adjuvant (Difco). A mouse (BALB/c, 5 weeksold, Japan SLC, Inc.) was immunized with the mixture three times, andthe spleen cells of the mouse were used in cell fusion using Sp2/0-Ag14mouse myeloma cells (Shulman, et al., 1978). The cells were cultured ina culture solution prepared by adding L-glutamine (0.3 mg/mL),penicillin G potassium (100 unit/mL), streptomycin sulfate (100 μg/mL),and Gentacin (40 μg/mL) to Dulbecco's Modified Eagle Medium (DMEM)(Gibco) and also adding fetal calf serum (JRH) thereto in an amount of10%. The cell fusion was performed by mixing immunized mouse spleencells with Sp2/0-Ag14 cells and adding polyethylene glycol solution(Sigma) to the mixture. The hybridomas were cultured in HAT-DMEM(serum-added DMEM containing 0.1 mM sodium hypoxanthine, 0.4 μMaminopterin, and 0.016 mM thymidine (Gibco)). Antibody production in theculture supernatant was verified by enzyme-linked immunosorbent assay(ELISA). Antibody production-positive cells were cultured in HT-DMEM(serum-added DMEM containing 0.1 mM sodium hypoxanthine and 0.16 mMthymidine) and were further continuously cultured in serum-added DMEM.

Example 3: Preparation of Monoclonal Antibody

A mouse (BALB/c, retired, Japan SLC, Inc.), inoculated with2,6,10,14-tetramethylpentadecane (Sigma) in advance, wasintraperitoneally inoculated with the cloned cells, and the ascites wascollected. The ascites was applied to a protein G column to purify amonoclonal antibody. The isotype of the produced monoclonal antibody wasidentified by Mouse Monoclonal Antibody Isotyping Reagents (Sigma).

Eventually, six clones of cells producing monoclonal antibodies againstP30 protein were obtained.

Reference Example 1: Production of Standard Bacterial Solution for Test

PPLO media were inoculated with standard strains of Mycoplasmapneumoniae M129 strain and FH strain, followed by culturing in anatmosphere of 5% CO₂ at 37° C. until the desired concentration wasobtained. The resulting culture solution was 10-fold serially dilutedwith a PPLO liquid medium until giving 100000-fold diluted solution. Thenumber of grown colonies in each diluted solution on the PPLO agarmedium was counted under a stereomicroscope to calculate the bacterialconcentration. The resulting culture solutions were used as bacterialsolutions for tests.

Comparative Example 1: Purification of Mycoplasma pneumoniae P1 Protein

A PPLO liquid medium was inoculated with a Mycoplasma pneumoniae M129strain, followed by culturing at 37° C. The resulting culture solutionwas centrifuged to collect the cells. P1 protein was purified from thecells in accordance with the method of Nakane, et al. (Journal ofBacteriology, 2011).

The resulting cells were washed with a PBS, pH 7.4, twice. The cellswere suspended in a PBS containing 1% CHAPS, and the suspension wascentrifuged. The resulting sediment was then dissolved in a PBScontaining 2% octylglucoside. The solution was centrifuged, and thesupernatant was collected. The resulting supernatant was subjected toammonium sulfate fractionation, followed by centrifugation. Theresulting sediment was dissolved in a PBS containing 0.3% Triton X-100,and the solution was purified by gel filtration column chromatographyusing Superdex 200. The fraction containing the purified protein wasanalyzed by SDS-page to confirm a single band at about 170 kDa. Thus,target P1 protein was obtained.

Example 4: Epitope Analysis of Anti-P30 Antibody

Amino acid sequences that are present in proline-rich regions onN-terminal side and thus may form epitopes were selected from the aminoacid sequence of P30 protein derived from Mycoplasma pneumoniae of SEQID NO: 1, and polypeptides having amino acid sequences of SEQ ID NOS: 3to 5 were synthesized. The synthesized polypeptides had sequences ofGMAPRPGMPPHP (SEQ ID NO: 3) of No. 178 to No. 189, GMAPRPGFPPQP (SEQ IDNO: 4) of No. 190 to No. 201 and GMAPRPGMQPPRP (SEQ ID NO: 5) of No. 250to No. 262 from the N-terminal of the amino acid sequence of SEQ IDNO: 1. Meanwhile, the same sequence as the sequence shown in SEQ ID NO:3 is present between No. 202 and No. 213 and between No. 226 to No. 237from the N-terminal, and also the same sequence as the sequence shown inSEQ ID NO: 4 is present between No. 214 and No. 225 and between No. 238and No. 249 from the N-terminal. That is, a plurality of amino acidsequences of SEQ ID NOS: 3 and 4 exist in P30 protein so as to providerepeating structures.

Peptides having amino acid sequences of SEQ ID NOS: 3 to 5 were added toa 96-well microplate and immobilized. As controls, M. pneumoniaepurified cells, the purified P30 protein prepared in Example 1,polypeptides having sequences of KRKEKRLLEEKERQEQLORIS (SEQ ID NO: 6) ofNo. 101 to No. 125 and AQQEEQQALEQQAAAEAHAE (SEQ ID NO: 7) of No. 126 toNo. 145 from the N-terminal of P30 protein, and the P1 protein ofMycoplasma pneumoniae prepared in Reference Example 1 were immobilized,and the reactivity with the monoclonal antibodies produced in Example 3was confirmed in the same manner as above.

Monoclonal antibodies produced in Example 3 were added to the microplateto which the peptides were immobilized at prescribed concentrations,followed by incubation at room temperature for 1 hour. Subsequently, thesolution in each well was sucked and removed. After washing, abiotin-labeled anti-mouse antibody was added for reaction. Afterincubation for 1 hour, the solution in each well was sucked and removed.After washing, avidin-labeled horse radish peroxidase was added forreaction. A 3,3′,5,5′-tetramethylbenzidine (TMBZ) solution was thenadded as a chromogenic substrate for reaction. The reaction was stoppedwith 2N sulfuric acid. The absorbance was measured with a microplatereader (Biorad) at a main wavelength of 450 nm. The results are shown inTable 1.

TABLE 1 Immobilized antigen SEQ ID SEQ ID SEQ ID P30 SEQ ID SEQ ID P1 M.pneumoniae NO: 3 NO: 4 NO: 5 protein NO: 6 NO: 7 protein purified cellsBLMP001 0.894 1.021 1.863 1.813 0.062 0.051 0.012 2.124 BLMP002 2.3010.541 0.721 1.851 0.051 0.026 0.025 2.052 BLMP003 0.781 2.012 0.6211.921 0.021 0.047 0.014 1.856 BLMP004 1.729 1.531 1.621 2.120 0.0420.031 0.037 1.741 BLMP005 0.062 0.051 0.043 1.613 1.593 0.042 0.0421.684 BLMP006 0.021 0.044 0.025 1.489 0.027 1.626 0.018 1.729

From the above results, the monoclonal antibody BLMP001 showed thestrongest reaction with the polypeptide having the amino acid sequenceof SEQ ID NO: 5. Also, it showed a reaction with the polypeptides havingsimilar sequences, namely, the polypeptide having the amino acidsequence of SEQ ID NO: 3 and the polypeptide having the amino acidsequence of SEQ ID NO: 4.

The monoclonal antibody BLMP002 showed a strong reaction with thepolypeptide having the amino acid sequence of SEQ ID NO: 3, and themonoclonal antibody BLMP003 showed a strong reaction with thepolypeptide having the amino acid sequence of SEQ ID NO: 4. They showeda reactivity with other polypeptides having the similar amino acidsequences, as the monoclonal antibody BLMP001 did.

The monoclonal antibody BLMP004 showed a reaction with all thepolypeptides having the amino acid sequence of SEQ ID NO: 3, SEQ ID NO:4 and SEQ ID NO: 5. It is assumed to be a monoclonal antibodyrecognizing a sequence common in the respective amino acid sequences.

The monoclonal antibodies BLMP001, BLMP002 and BLMP003 showed a strongreaction with the Mycoplasma pneumoniae purified cells and the purifiedP30 protein but showed no reaction with the purified P1 protein as acontrol.

Therefore, it was confirmed that the monoclonal antibody BLMP001 is anantibody recognizing a polypeptide having the amino acid sequence of SEQID NO: 5 in the P30 protein, the monoclonal antibody BLMP002 is anantibody recognizing a polypeptide having the amino acid sequence of SEQID NO: 3, and the monoclonal antibody BLMP003 is an antibody recognizinga polypeptide having the amino acid sequence of SEQ ID NO: 4.

Example 5: Production of Immunochromatographic Test Strip Using Anti-P30Antibody (1) Preparation of Anti-P30 Antibody

Mice were intraperitoneally inoculated with monoclonal antibody BLMP001producing cells or monoclonal antibody BLMP004 producing cells preparedin Example 3, and the ascites obtained from each mouse was purified withprotein G by an ordinary method to obtain IgG which was used as ananti-P30 antibody.

(2) Preparation of Platinum-Gold Colloidal Particle Solution

Glassware to be used was all washed with aqua regia. Ultrapure water(390 mL) was boiled in a flask, and an aqueous chloroauric acid solution(30 mL, 1 L of the aqueous solution contains 1 g of gold, manufacturedby Katayama Chemical Industries Co., Ltd.) was added to the boilingwater. A 1 wt % aqueous sodium citrate solution (60 mL) was then addedto the flask, and after 6 min and 45 sec, an aqueous chloroplatinic acidsolution (30 mL, 1 L of the aqueous solution contains 1 g of platinum,manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto.At 5 min after the addition of the aqueous chloroplatinic acid solution,a 1 wt % aqueous sodium citrate solution (60 mL) was added thereto,followed by reflux for 4 hours to obtain a platinum-gold colloidalsuspension.

(3) Preparation of Platinum-Gold Colloid-Labeled Anti-P30 AntibodySolution

The monoclonal antibody BLMP001 obtained in the above section (1) wasused as an anti-P30 antibody to be labeled with the platinum-goldcolloid, and the labeling with the platinum-gold colloid was performedby the following procedure.

The anti-P30 antibody (1 μg in terms of protein weight, hereinafter, theweight of an antibody in terms of protein weight is simply shown by anumerical value of weight obtained by gravimetric analysis of thepurified protein) and the platinum-gold colloidal solution (1 mL)described in the above section (2) were mixed, and the mixture was leftto stand at room temperature for 2 minutes to allow all of the antibodyto bind to the surfaces of the platinum-gold colloidal particles. Anaqueous 10% bovine serum albumin (hereinafter, referred to as “BSA”)solution was then added thereto at a final concentration of 1% in theplatinum-gold colloidal solution to block all of the residual surfacesof the platinum-gold colloidal particles with BSA. Thus, a platinum-goldcolloid-labeled anti-P30 antibody (hereinafter, referred to as“platinum-gold colloid-labeled antibody”) solution was prepared. Thissolution was centrifuged (5600×G, for 30 min) to precipitate theplatinum-gold colloid-labeled antibody, and the supernatant was removedto obtain a platinum-gold colloid-labeled antibody. This platinum-goldcolloid-labeled antibody was suspended in a 50 mM tris hydrochloric acidbuffer solution (pH 7.4) containing 10% saccharose, 1% BSA, and 0.5%Triton-X 100 to obtain a platinum-gold colloid-labeled antibodysolution.

(4) Production of Immunochromatographic Test Strip Detecting P30 Proteinof Mycoplasma pneumoniae

(4-1) Capturing Zone of Complex of P30 Protein of Mycoplasma pneumoniaeand Gold Colloid-Labeled Antibody

An elongated strip-shaped nitrocellulose membrane with a size of 5 mmwidth and 36 mm length was provided as a membrane carrier 3 forchromatographic development of a chromatographic medium. 0.5 μL of asolution containing 1.0 mg/ml anti-P30 antibody was applied in a linearform to a position of 7.5 mm from the end on the starting point side ofthe chromatographic development of the membrane carrier 3 forchromatographic development. It was dried at room temperature, to form acapturing zone 31 for capturing a complex of the P30 protein and theplatinum-gold colloid-labeled antibody. The applied anti-P30 antibodywas the monoclonal antibody BLMP004 obtained in the above section (1).

(4-2) Platinum-Gold Colloid-Labeled Antibody-Impregnated Member

A strip-shaped glass fiber nonwoven fabric with a size of 5 mm×15 mm wasimpregnated with 37.5 μL of the platinum-gold colloid-labeled antibodysolution, and then dried at room temperature, to obtain a platinum-goldcolloid-labeled antibody impregnated member 2.

(4-3) Preparation of Immunochromatographic Test Strip

In addition to the membrane carrier 3 for chromatographic developmentand the labeled antibody-impregnated member 2, a cotton fabric as thesample-receiving member 5 and a filter paper as the absorbing member 4were prepared. Then, a chromatographic test strip which was the same asin FIGS. 1A and 1B was prepared using these members.

(5) Test

The cultured bacterial solutions of M129 strain and FH strain ofMycoplasma pneumoniae were diluted with a sample extraction solutioninto a prescribed concentration to prepare each test sample. The testsample (100 μL) was dropwise added with a micropipette to the samplereceiving member 5 of the test strip described in the above section (4)for chromatographic development by being left to stand at roomtemperature for 15 minutes. The captured amount of the complex of theP30 protein and the platinum-gold colloid-labeled antibody captured bythe capturing zone 31 was observed with the naked eye. The capturedamount was determined by evaluating the degree of blacking, which isproportional to the amount, with the naked eye and classified into thefollowing five stages: − (no blacking), ± (slight blacking), + (clearblacking), ++ (noticeable blacking), and +++ (extremely noticeableblacking). The cultured bacterium solution of M. genitalium was used asa negative control at a predetermined concentration.

Table 2 shows the results. As obvious from Table 2, high reactivity wasshown with the two strains of Mycoplasma pneumoniae, and negativity wasshown in all the tested concentrations against M. genitalium as thenegative control. It was revealed that Mycoplasma pneumoniae can bedetected with high sensitivity and high accuracy by theimmunochromatographic assay using the two types of anti-P30 antibody.

TABLE 2 M. pneumoniae M. pneumoniae M. M129 strain FH strain genitaliumConcentration Blank − − − (CFU/ml) 1 × 10⁴ ± ± − 1 × 10⁵ + ±~+ − 1 × 10⁶++ + − 1 × 10⁷ +++ ++ −

Example 6: Comparative Reactivity Test of P30 Protein-DetectingImmunochromatographic Test Strip and P1 Protein-DetectingImmunochromatographic Test Strip

The cultured bacterial solutions of M129 strain and FH strain ofMycoplasma pneumoniae prepared in Reference Example 1 were diluted witha sample extraction solution into a prescribed concentration to prepareeach test sample. The test sample (100 μL) was dropwise added with amicropipette to the sample receiving member 5 of eachimmunochromatographic test strip for chromatographic development bybeing left to stand at room temperature for 15 minutes. The complex ofthe antigen and the platinum-gold colloid-labeled antibody captured bythe capturing zone 31 was observed with the naked eye to determine theresult. The captured amount was determined by evaluating the degree ofblacking, which is proportional to the amount, with the naked eye andclassified into the following five stages: − (no blacking), ± (slightblacking), + (clear blacking), ++ (noticeable blacking), and +++(extremely noticeable blacking). As the conventional method, acommercially available reagent for detecting P1 protein of Mycoplasmapneumoniae was used. The results are shown in Table 3.

TABLE 3 M. pneumoniae M. pneumoniae M129 strain FH strain M. genitaliumPresent Conventional Present Conventional Present Conventional inventionmethod invention method invention method Concentration Blank − − − − − −(CFU/ml) 1 × 10⁴ ± − ± − − − 1 × 10⁵ ±~+ − + − − − 1 × 10⁶ ++ ± ++ ± − ±1 × 10⁷ +++ ±~+ +++ + − ±

As obvious from Table 3, the P30 protein-detecting immunochromatographictest strip prepared in Example 5 of the present invention showednoticeable blacking for 1×10⁷ CFU/mL of M129 strain and 1×10⁶ CFU/mL ofFH strain and showed clear blacking for 1×10⁶ CFU/mL of M129 strain and1×10⁵ CFU/mL of FH strain.

In contrast, the conventional method showed clear blacking for 1×10⁷CFU/mL or more of M129 strain and 1×10⁷ CFU/mL of FH strain.

As obvious from the results shown in Table 3, the results of comparisonof the bacterial concentrations of test samples at which the same degreeof blacking was shown demonstrated that the detection sensitivity of theP30 protein-detecting immunochromatographic test strip prepared inExample 5 of the present invention was about 100-times higher for M129strains and also 100-times higher for FH strains, compared with thesensitivity of the P1 protein-detecting immunochromatographic teststrip. In addition, the conventional method showed a slightcross-reactivity with Mycoplasma genitalium, and non-specific blackingwas observed. No cross-reactivity was observed on the P30protein-detecting immunochromatographic test strip of the presentinvention.

The results described above demonstrated that the P30 protein-detectingimmunochromatographic test strip using the anti-P30 antibody of thepresent invention can detect Mycoplasma pneumoniae with high sensitivityand specificity.

Example 7: Detection of Mycoplasma pneumoniae from Throat Swab

Throat swabs were collected from 20 patients clinically suspected ofinfection with Mycoplasma pneumoniae with sterilized cotton swabs. Thethroat swabs were each verified as to whether Mycoplasma pneumoniae waspresent in the throat swab or not by the nucleic acid amplification testreported by National Institute of Infectious Diseases, Japan. Based onthe results, sixteen samples (positive samples) in which the presence ofMycoplasma pneumoniae was confirmed and four samples (negative samples)in which the gene was not detected were selected from the collectedthroat swabs. The selected throat swabs were prepared as test samples.The test samples were subjected to detection of Mycoplasma pneumoniaeusing the P30 protein-detecting immunochromatographic test strip of thepresent invention. A commercially available reagent detecting P1 proteinof Mycoplasma pneumoniae was used as a conventional method.

The test sample (100 μL) was dropwise added with a micropipette to thesample receiving member 5 of the immunochromatographic test stripprepared in Example 5 for chromatographic development by being left tostand at room temperature for 15 minutes. The complex of the antigen andthe platinum-gold colloid-labeled antibody captured by the capturingzone 31 was observed with the naked eye to determine the result. Thecaptured amount was determined by evaluating the degree of blacking,which is proportional to the amount, with the naked eye and classifiedinto the following five stages: − (no blacking), ± (slight blacking), +(clear blacking), ++ (noticeable blacking), and +++ (extremelynoticeable blacking). Table 4 shows the results of the test.

TABLE 4 Nucleic acid Present Conventional amplification test Sample No.invention method (PCR method) 1 ++ + + 2 ++ + + 3 +++ + + 4 ++ ± + 5 ++± + 6 + ± + 7 − ± − 8 − ± − 9 ± − + 10 ++ + + 11 − − + 12 + ± + 13 + ± +14 − − − 15 ± − + 16 + − + 17 ± − + 18 − − − 19 + ± + 20 + ± +

As obvious from Table 4, the comparison between the detection method ofthe present invention and the nucleic acid amplification test shows thatthe detection method of the present invention exhibited the highconcordance rate, namely, a positive concordance rate of 93.8%, anegative concordance rate of 100.0%, and a total concordance rate of95.0%, confirming that the detection method of the present invention canperform the detection with an accuracy equivalent to the nucleic acidamplification test. It is also shown that the detection method of thepresent invention is a detection method having high detectionsensitivity and specificity, compared with the conventional method.

From the above results, it was confirmed that the immunochromatographictest strip of the present invention can detect Mycoplasma pneumoniaefrom throat swabs with high sensitivity and high accuracy.

INDUSTRIAL APPLICABILITY

The present invention provides a method and a kit for detectingMycoplasma pneumoniae, comprising an immunoassay using an antibodyrecognizing a specific epitope of P30 protein of Mycoplasma pneumoniae,and makes it possible to rapidly and specifically diagnose infectionwith Mycoplasma pneumoniae without need of any specialized equipment orskill, compared with the conventional method.

DESCRIPTION OF SYMBOLS

-   -   1 Adhesive sheet    -   2 Impregnated member    -   3 Membrane carrier    -   31 Capturing zone    -   32 Control capturing zone    -   4 Absorbing member    -   5 Sample receiving member

1. A method of detecting Mycoplasma pneumoniae, comprising animmunoassay using an antibody against P30 protein of Mycoplasmapneumoniae, the antibody being an antibody against an epitope of P30protein located in any one of amino acid sequences of SEQ ID NOS: 3 to5.
 2. The detecting method according to claim 1, wherein the antibody isa monoclonal antibody.
 3. A method of detecting Mycoplasma pneumoniae,comprising a sandwich immunoassay using first and second antibodiesagainst P30 protein of Mycoplasma pneumoniae, wherein at least one ofthe first and second antibodies is an antibody against an epitope of P30protein located in any one of amino acid sequences of SEQ ID NOS: 3 to5.
 4. (canceled)
 5. The detecting method according to claim 3, whereinat least one of the first and second antibodies is a monoclonalantibody.
 6. The detecting method according to claim 5, wherein one ofthe first and second antibodies is immobilized in a carrier. 7.(canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. An immunoassaykit for Mycoplasma pneumoniae, comprising at least an antibody againstP30 protein of Mycoplasma pneumoniae, wherein the antibody is anantibody against an epitope of P30 protein located in any one of aminoacid sequences of SEQ ID NOS: 3 to
 5. 12. A sandwich immunoassay kit forMycoplasma pneumoniae, comprising at least first and second antibodiesagainst P30 protein of Mycoplasma pneumoniae, wherein at least one ofthe first and second antibodies is an antibody against an epitope of P30protein located in any one of amino acid sequences of SEQ ID NOS: 3 to5.
 13. The sandwich immunoassay kit according to claim 12, wherein thesandwich immunoassay is an ELISA or immunochromatographic assay.
 14. AMycoplasma pneumoniae-detecting immunochromatographic test strip,comprising at least first and second antibodies against P30 protein ofMycoplasma pneumoniae and a membrane carrier, wherein the first antibodyis previously immobilized at a predetermined position of the membranecarrier so as to form a capturing zone; and the second antibody islabeled with an appropriate labeling substance and is provided at aposition separated from the capturing zone so as to bechromatographically developed in the membrane carrier, wherein at leastone of the first and second antibodies is an antibody against an epitopeof P30 protein located in any one of amino acid sequences of SEQ ID NOS:3 to
 5. 15. The immunochromatographic test strip according to claim 14,wherein at least one of the first and second antibodies is a monoclonalantibody.
 16. The immunochromatographic test strip according to claim15, wherein the second antibody is labeled with any one of a colloidalmetal, a latex, and a fluorescent substance.
 17. Theimmunochromatographic test strip according to claim 16, wherein themembrane carrier is a nitrocellulose membrane.
 18. (canceled) 19.(canceled)
 20. The detecting method according to claim 2, which exhibitsa sensitivity sufficient for detecting Mycoplasma pneumoniae strain M129in a culture having a concentration of 1×10⁶ CFU/ml of the M129 strain.21. The detecting method according to claim 5, wherein the first andsecond antibodies, which may be the same or different from each other,are each a monoclonal antibody against an epitope of P30 protein locatedin any one of amino acid sequences of SEQ ID NOS: 3 to 5, said detectingmethod exhibiting a sensitivity sufficient for detecting Mycoplasmapneumoniae strain M129 in a culture having a concentration of 1×10⁶CFU/ml of the M129 strain.
 22. The immunoassay kit according to claim11, wherein the antibody is a monoclonal antibody, said immunoassay kitexhibiting a sensitivity sufficient for detecting Mycoplasma pneumoniaestrain M129 in a culture having a concentration of 1×10⁶ CFU/ml of theM129 strain.
 23. The sandwich immunoassay kit according to claim 12,wherein the first and second antibodies, which may be the same ordifferent from each other, are each a monoclonal antibody against anepitope of P30 protein located in any one of amino acid sequences of SEQID NOS: 3 to 5, said sandwich immunoassay kit exhibiting a sensitivitysufficient for detecting Mycoplasma pneumoniae strain M129 in a culturehaving a concentration of 1×10⁶ CFU/ml of the M129 strain.
 24. Theimmunochromatographic test strip according to claim 15, wherein thefirst and second antibodies, which may be the same or different fromeach other, are each a monoclonal antibody against an epitope of P30protein located in any one of amino acid sequences of SEQ ID NOS: 3 to5, said immunochromatographic test strip exhibiting a sensitivitysufficient for detecting Mycoplasma pneumoniae strain M129 in a culturehaving a concentration of 1×10⁶ CFU/ml of the M129 strain.
 25. Thedetecting method according to claim 21, wherein the sandwich immunoassayis an ELISA or immunochromatographic assay.